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GANDHI SANKET J.1st YEAR M.PHARMDept. of pharmacology R. C. Patel Institute of Pharmaceutical Education and Research

ACUTE ORAL

CARCINOGENICITY

CHRONIC

TOXICITY STUDY

OECD GUIDELINES

CONTENTS2

INTRODUCTION TO TOXICOLOGY

OECD GUIDELINE FOR ACUTE ORAL TOXICITY

LD50 LD50/LC50 Methods to calculate LD50 Limitation of LD50 How OECD GUIDELINES More Humane? Alternatives to use of Animal in AOT

Description of Whole AOT Guidelines along with Its Sighting Study (Guidelines no. 401,420,423,425)

OECD GUIDELINES FOR CARCINOGENICITY (451) (Principle ,Housing Feeding,Objectives, Procedure,Result,DiscussIon)

OECD GUIDELINES FOR CHRONIC TOXICITY (452) (Principle ,Housing Feeding,Objectives, Procedure,Result,DiscussIon)

WHAT IS TOXICOLOGY? Toxicology is the scientific study of adverse effects that occur in living organisms due to chemicals. It involves observing and reporting symptoms, mechanisms, detection and treatments of toxic substances, in particular relation to the poisoning of humans.3

ADVERSE DRUG EFFECTSAny undesirable &/ or unintended effects of drug,

1. Predictable (type A reactions)2. Non-predictable (type B reactions)

SIDE EFFECTS Unwanted but often unavoidable effects at therapeutic doses. SECONDARY EFFECTS

- Indirect consequences of primary action of drug.

TOXIC EFFECTS Are results of excessive pharmacological effect of drug due to over dosage or prolonged use. 4(Barar,2005; Ghosh, 2005)

ANIMAL TOXICITY TESTSAcute toxicity 14 days

Sub-acute (repeated doses) toxicity 28 days

Sub-chronic toxicity 3 months

Chronic toxicity 6 months to 2

Special toxicity e.g. Carcinogenicity

5(Curtis,2003;Ghosh,2005)

ACUTE ORAL TOXICITY

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ACUTE TOXICITY SYMPTOMSTremor

Salivation

MusclespasmConvulsion LacrimationWeight loss DiarrhoeaAlteredRespiration Loss of righting reflex7(Ghosh., 2005)

WHAT IS LD50?

LD50 represents the individual dose required to kill 50 percent of a population of test animals. It is an index determination of medicine and poisons virulence. lower the LD50 dose, the more toxic the pesticide.

WHAT IS LC50?

The concentrations of the chemical in air that kills 50% of the test animals during the observation period is the LC50value. Other durations of exposure (versus the traditional 4 hours) may apply depending on specific laws.

8(P.A. Botham et al. 2004)

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LD50/LC50

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METHODS TO DETERMINE LD50 Karbers method

Miller and Tainter method

Lorkes method

Fixed dose method

UP and down method

10OECD,1987; Ghosh, 2005)

KARBER`S METHOD This method is also known as ARITHMATIC method. Simplest & rapid. It does not involve any plotting of dose-response curve. It is useful method particularly when number of animals is small. The interval mean of the no. of dead in each group of animals was used as well as the differences between doses for the same interval. The number of animal in every group must be equal.

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LD50 CALCULATION BY KARBER`S METHODGroupDoseMg/kgNo. of animalDose difference (a)deadMean mortality (b)Product(a.b)16410-0--2711072173811010433049010996.558.551001010109.595

LD50 = 100 {a.b/n}

n is the number of animals12

MILLER AND TAINTER METHOD Also called as Graphical method Simple & accurate enough. The observed % mortality is converted into probit ( deviation from the mean) by referring to the table. Percentage dead for 0% & 100% are corrected. The probit value thus obtained are plotted against log dose. And then dose corresponding to probit 5 (50%) is found out. The LD50 may be determined from the graph if the line is straight enough.13

LD50 CALCULATION BY GRAPHICAL METHODGroupDoseMg/kgLog doseDead/total% Deadcorrected % dead probit 1 641.810/10 02.5- 2 711.852/10 20 204.16 3 811.914/10 40 404.75 4 901.959/10 90 906.28 5 1002.0010/10 100 97.5-

corrected formula for 0 % = 100(0.25/n)

corrected formula for 100% = 100{(n 0.25) /n}14

%0 1234567890-2.67 2.953.123.253.363.453.523.593.66103.723.773.823.873.923.964.014.054.084.12204.164.194.234.264.294.334.364.394.424.45304.484.504.534.564.594.614.644.674.694.72404.754.774.804.824.854.874.904.924.954.97505.005.035.055.085.105.135.155.185.205.23605.255.285.315.335.365.395.415.445.475.50705.525.555.585.615.645.675.715.745.775.81805.845.885.925.955.996.046.086.136.186.23906.286.346.416.486.556.646.756.887.057.33

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TRANSFORMATION OF PERCENTAGES TO PROBITS (GHOSH,. 2005)15

GRAPHICAL METHOD

-------------------------------------.....1.81.92.034567--------Log dose PROBIT16

LORKES METHOD Phase 1: 3 groups of mice are prepared .one dose was given to each group i.p The treated mice were monitored for 24hr for mortality and general behavior .

Phase 2: After 24 hr 3-4 groups of one mouse were given doses based on the findings of phase 1. I.p . The mice were again monitored for 24 hrs .The geographic mean of least dose that killed mice and the highest dose that did not kill mice was taken as the median lethal dose

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ALTERNATIVE METHOD (1) FIXED DOSE PROCEDURE

This method does not use death as an end points instead it uses the observation of clear signs of toxicity developed at one of a series of fixed dose levels to estimate the LD50

(2) UP AND DOWN METHOD (Staircase method)

Two mice were injected with a particular dose and observed for a period of 24 hrs for any mortality .

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The subsequent doses were then increased by a factor 3.2 if the dose was tolerated ,or if not then decreased by a factor of 0.5. The max. Non lethal and the min lethal doses were thus determined using only 15 mice.

A final and more reliable LD50 assay was planned using at least 3 or 4 dose levels within the range of max non lethal and min doses

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LD50 LIMITATIONSLD50is somewhat unreliable and results may vary greatly between testing facilities due to factors such as the genetic characteristics of the sample population, animal species tested, environmental factors and mode of administration

There can be wide variability between species as well; what is relatively safe for rats may very well be extremely toxic for humans (cf.paracetamol toxicity), and vice versa. For example, chocolate, comparatively harmless to humans, is known to betoxic to many animals. When used to testvenomfrom venomous creatures, such assnakes, LD50results may be misleading due to the physiological differences between mice, rats, and humans.20

HOW OECD GUIDELINES FOR ACUTE TOXICITY MORE HUMANE ?A humane endpoint can be defined as the earliest indicator in an animal experiment of severe pain, severe distress, suffering, or impending death.OECD Test Guidelines do not require death as an endpoint. Animals humanely killed during the test will be regarded as dosage-dependent deaths.Three alternative test methods (Guidelines 420, 423, and 425) to the traditional acute oral toxicitytest have been adopted by the OECD. One of these, the Fixed Dose Procedure (Guideline 420).21

CONTINUERefinement of the traditional acute oral test in that it requires fewer, but fixed, dosage groups to be tested, and thus fewer animals. It also employs non-lethal endpoints to determine the toxicity of the test substance.

Two other methods, the Acute Toxic Class Method (Guideline 423) and the Up-and-Down Procedure (Guideline 425), use impending death as the only endpoint.

Series on Testing and Assessment: Testing for Human Health in that guidline no. 19 contains Guidance document on the recognition, assessment and use of clinical signs as humane endpoints

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CONTINUEGUIDLINESNUMBER OF ANIMAL PER TEST NUMBER OF DEATH PER TESTAIM401Up to 25Up to 12-4205-71Endpoint-evidenttoxicity423Average 72-3-4256-92-3Stopping criteria tolimit number ofanimals used

Animal welfare considerations,All three alternatives contain requirement to follow OECD Document on Humane Endpoints.23

SOME ALTERNATIVES TO THE USE OF ANIMALS IN TESTING in vitro (test tube) test methods and models based on human cell and tissue cultures.computerized patient-drug databases and virtual drug trials.computer models and simulations.stem cell and genetic testing methods.non-invasive imaging techniques such as MRIs and CT Scans.microdosing (in which humans are given very low quantities of a drug to test the effects on the body on the cellular level, without affecting the whole body system).

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ICCVAM

The need for alternatives to the traditional use of animals in toxicity testing was officially recognized by the U.S. government in 1993 with passage of the NIH Reauthorization Act. Requirements under the Act lead to the establishment of committee called the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM). To Providing guidance to test method developersTo Induce use of alternative tests that encourage the reduction, refinement, or replacement of animal test methodsEvaluating recommendations from expert peer reviews of alternative toxicological test methods

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OECD GUIDELINES FOR ACUTE ORAL TOXICITYNumberTitleOriginal adoptionNumber of updatesMost recently updated401Acute oral toxicity- conventional acute toxicity test12 may 1981 1Date of deletion: 20 December 2002420Acute oral toxicity- fixed dose procedure17 July 1992 117 December 2001423Acute oral toxicity- acute toxic class method22 march 1996 117 December 2001425Acute oral toxicity- up and down procedure21 September 1998 223 March 2006

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DESIGN OF ACUTE TOXICITY14 days study.Study on at least two species.One rodent mice/rat.One non rodent usually rabbit.Dose administered orally & parenterally.Various dose levels to groups of both sexes. Dose selection such that causing less than 50% but not 0% and more than 50% but not 100% mortality.

27(Ghosh,2005)

ACUTE ORAL TOXICITY OECD GUIDELINES NO.401 (CONVENTIONAL ACUTE TOXICITY METHOD )In a study of toxic characteristics of substance, acute oral toxicity testing is initial step.

Gives information on health hazards.

Test substance administered orally, in graduated doses to several groups of experimental animals.

One dose used per group.

28(OECD,1987)

CONTINUEAt least 5 rodents at each dose level of same sex are used.

Observations for effects & death are made.

After completion of study in one sex, study in another sex is carried out.

Studies suggested in rodents but can be adopted for studies in non-rodents.

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ACUTE ORAL TOXICITY GUIDLINE NO. 420 (FIXED DOSE PROCEDURE)New approach in 1984 by British toxicology society based on administration of series of fixed dose levels.Instead of death, clear signs of toxicity to animals as end point.Adopted as 1st alternative to conventional acute toxicity test.Testing in 1 sex usually females is considered sufficient.Uses fewer animals.

30(OECD, 2001)

CONTINUEReproducible procedure. Causes less suffering to the animals.Uses only moderately toxic doses, doses expected to be lethal should be avoided.

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PROCEDURE Procedure involves 2 step study

1- sighting study 2-main study

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FLOW CHART FOR SIGHTING STUDY

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FLOW CHART FOR MAIN STUDY

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ACUTE ORAL TOXICITY GUIDLINE NO.423 (ACUTE TOXIC CLASS METHOD)No sighting study.3 animals of single sex per step. On avg. 2-4 steps may be necessary to allow judgment on the acute toxicity of the test substance. Not intended to allow the calculation of precise LD50 .Death of a proportion of animals as the major end point (response).Ld50 cut off values are indicated .

37(OECD, 2001)

PRINCIPLEStepwise procedure with the use of minimum no. of animals per step. Substance administered orally to 2 groups of animals at defined doses .3 animals per step of single sex (normally females).Compound related mortality determines the next step. Report

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FLOW CHART

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CONTINUE

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CONTINUE

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ACUTE ORAL TOXICITY GUIDLINE NO.425 (UP AND DOWN METHOD)Up and down testing approach was 1st described by Dixon and Mood. Bruce in 1985 proposed to use it for acute toxicity determination of chemicals .Estimates confidence intervals for LD50 .

42(OECD2006)

CONTINUE In procedure (main test ) 1-animal dosed at a time, at minimum of 48 hrs interval.Suggested starting dose is 175 mg/kg or can be selected from 1.75, 5.5, 17.5, 55,175,550,2000mg/kg .Animal receives 1st dose a step below the level of the best estimate of LD50 .

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CONTINUE Depending upon the outcome for the previous animal, the dose for the next animal is adjusted up or down.5 reversal in 6 consecutive animals when obtained test is terminated.No. of animals limited to 15. Report

AOT 425 START PROG.44

COMPARISON OF GUIDELINES OF LD50

No 401420423425Sighting studyAbsentPresent AbsentAbsentPreferred dose 2 to 5000mg/kg per animal5 to 5000 mg/kg per animal5 to 5000 mg/kg per animal1st animal dose less than estimated Ld 50 outcome by factor 3.2 Drug effectDose causing high mortalityDose causing evident toxicity Dose causing mortalityStopped if no reversal occur by reaching selected upper limit Grouping of animal 5 animals per groupGroup of 5 animal of 1 sex3 animals per group1 animal per groupEstimation of end pointUp to 12 animal expected to die 1 animal expected to die2,3 animal expected to die2,3 animal expected to dieLimitationIt used high animal mortality as endpoint, at high dose, so this method require reduction or refinement.It used evident toxicity as endpoint instead of death dose level close to lethal dose not always obtainedFinding of a delayed death may require additional lower dose level to be used No limitation

45(R. L. LIPNICK et al.1995)

LIMITATION OF METHODS USED FOR TOXICITY STUDY Indicated that all are likely to perform poorly for chemicals with shallow dose-response slopes Because Guideline 420 uses evident toxicity as an endpoint instead of death.Unusually test substances may cause delayed deaths (5 days or more after test substance administration) mostly in case of guidline no.425 However, both in Guideline 420 and 423, the finding of a delayed death may require additional lower dose levels to be used or a study to be repeated.46

CARCINOGENICITY STUDYThe test substance is administered daily in graduated doses

Observed closely for signs of toxicity and for the development of neoplastic lesions.

Died or are killed animals are necropsied and at the conclusion surviving animals are also killed and necropsied.

PRINCIPLEROUTES OF ADMINISTRATION

45145147www.OECD.org

OBJECTIVES OBJECTIVES48

HOUSING AND FEEDING 49

ANIMAL TO BE USED,Rodents/non-rodents used

12 months study for rodents

90 days study for non-rodents

Mostly mice are used due to short life-span and susceptibility

Healthy animals, acclimated to laboratory conditions for 7 days and not been subjected to previous experimental procedures

the weight variation of animals used should not exceed 20 % of the mean weight

Animals should be randomly assigned

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DOSE GROUP AND DOSAGE

51 3 Dose levelsshort-term repeated doseToxicological existing dataToxicokinetics existing dataRange finding studiesHighest dose level to identify the principal target organs and toxic effects (avoid severe toxicity, morbidity, or death)STUDY DURATION

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Observations53

RESULT

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DISCUSSION

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CHRONIC TOXICITY STUDYThe test substance is administered daily in graduated doses to several groups of experimental animals for a period of 12 months During the period of administration the animals are observed closely for signs of toxicity. Animals which die or are killed during the test are necropsied and, at the conclusion of the test, surviving animals are also killed and necropsied.452452PRINCIPLEROUTES OF ADMINISTRATION

56www.OECD.org

PREPARATION OF DOSE AND DURATION OF STUDYThe test substance is normally administered orally, by gavage or via the diet or drinking water.When the test substance is administered by gavage to the animals this should be done using a stomach tube or a suitable intubation cannula, at similar times each day.Normally the volume should be kept as low as practical, and should not exceed 1 ml/100g body weight, except in the case of aqueous solutions where 2 ml/100g body weight may be used.While this Test Guideline primarily is designed as a 12 month chronic toxicity study, the study design, also allows for and can be applied to either shorter (e.g. 6 or 9 months) or longer (e.g., 18 or 24 months)

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parametersBody weight, food/water consumption and food efficiency. (At least once a week for the first 13 weeks)Haematology and clinical biochemistry (10 females and 10 males)Total erythrocyte ,leucocytes and platelets count, erythrocytes morphology ,glucose ,urea , creatinine albumine ,total protiens count ,total cholesterol count, total bile level , presence of enzymesAppearance,Volume,Specific gravity,pH,Bilirubine,Glucose,Occult bloodUrine analysis59

Survival data; urinalysis tests Outcome of any investigations of neurotoxicity or immunotoxicity Terminal body weight; organ weights Necropsy findings; A detailed description of all treatment-related histopathological findings Absorption data if available Body weight/body weight changes Toxic response data by sex and dose level, including signs of toxicity Duration of clinical observations Ophthalmological examination Haematological tests Clinical biochemistry tests

RESULT60

General considerations such as housing, feeding, dose preparation, route of drug administration, histopathology, pathology, data collection, analyzing of data and results submission same as for OECD-451.61